Re: [Histonet] Intestinal Tissue Questions

From:

Geoff McAuliffe

Hi Wendy:
wbedale@biochem.wisc.edu wrote:
>Hi to all of you,
>
>I'm quite new to histology and have a few basic questions.
>
>Here's what I'm doing:
>
>Tissue: mouse intestine; small and large intestine is harvested, cut
>open longitudinally, and rinsed with PBS prior to fixation; 3 cm lengths
>are fixed.
>Fixation: 10% formalin
>Processing and embedding: manual processing, paraffin embedding, 4-6
>micron sections
>Staining: H&E staining
>
>Two questions:
>
>1. After fixation, is it better to store my tissue (for ~1 month or so)
>in fixative, or in 70% ethanol?
>
How long are you fixing? 48 hours is a minimum, a week is probably
better. Formalin works slowly. I don't know exactly what you are looking
for but I would try to standardize my processing schedule so all tissues
were treated for similar time periods. Storage in 70% is OK, but some
cytoplasmic components may be extracted. I would just go ahead and embed
rather than store in 70% for more than a week. Someone has certainly
studied this, but back in the 1950's, or even earlier.
>2. I'm attempting to get transverse sections of the intestine; sometimes
>I notice that my tissue ends up inside out (mucosa facing outward). I
>think it may happen when I use a razor blade to cut the tissue
>longitudinally vs. using scissors to make the longitudinal incision, and
>am going to test that. Has anyone had any experience with this?
>
Smooth muscle (in the muscularis externa) does this. If you want nice
cross sections cut the intestine open and pin it out flat on a piece of
cork sheet (Home Depot) or dental baseplate wax (see your dentist, much
cheaper than a supply house) using insect pins (#2 is a good size) while
it is fixing. You can turn the cork/was sheet upside down in a jar of
fixative. BETTER: don't cut it lengthwise, use a syringe or a pasteur
pipet to wash the food, etc out of the lumen then tie off both ends with
suture material/dental floss/thread leaving some fix in the lumen,
enough to fill the gut but not stretch it out too much. Make a sausage,
so to speak. After fixation, cut off the tied parts and you will have a
nice, round piece of gut. Works better on small intestine than on large,
more substantial wall in the small gut.
Call me on the phone for a method to "jelly roll" the whole gut.
Geoff (who worked on the small intesting during the last century)
>
>Thanks for any advice.
>
>Wendy
>
>Wendy Bedale, Ph.D.
>Assistant Scientist
>Department of Biochemistry
>University of Wisconsin-Madison
>433 Babcock Dr.
>Madison, WI 53706
>608-262-3099 ext. 3266
>wbedale@biochem.wisc.edu
>
>
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>
>
--
--
**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029
mcauliff@umdnj.edu
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